Evaluation in vitro of the virulence of two entomopathogenic heterorhabditid nematodes in the control of Stomoxys calcitrans (Diptera: Muscidae) larvae in byproducts of the sugar and alcohol industry

Abstract Stomoxys calcitrans causes losses to livestock, mainly to cattle. This study aimed to determine the pathogenic potential of Heterorhabditis bacteriophora HP88 and H. baujardi LPP7 against S. calcitrans larvae after being exposed to byproducts of the sugar and alcohol industry. The efficacy of EPNs on stable fly larvae was evaluated in bioassays with vinasse at three temperatures (16, 25 and 35 °C) and concentrations (0, 50 and 100%), as well as in relation to larva age (4, 6 and 8 days) in filter cake and EPNs concentration (100, 300 and 500 IJs/larva) in sugarcane bagasse. H. bacteriophora showed higher efficacy than H. baujardi at all temperatures. Vinasse did not have a negative effect on the virulence of H. bacteriophora. The age of fly larvae did not affect the mortality rates caused by the EPNs. In bagasse, H. bacteriophora presented higher mortality rates than the control group. It is concluded that EPNs can be a potential component in integrated strategies of stable fly control and outbreak prevention in areas of sugar and alcohol production.


Introduction
Stomoxys calcitrans, commonly called the stable fly, is a hematophagous dipteran that parasitizes several animal species, including cattle, goats, equines, pigs, and even humans. Its geographical distribution is wide, and its greatest population increase occurs during the hottest months of the year (Bittencourt & Moya-Borja, 2000). Parasitism by the stable fly poses a serious problem in raising livestock in several regions of Brazil. The country has suffered estimated economic losses of USD 335.5 million (Grisi et al., 2014). Several materials can be used to develop immature stages of S. calcitrans, particularly fermenting organic matter (Lysyk et al., 1999). The cultivation and generation of byproducts of the sugarcane industry provide a favorable environment for the proliferation of S. calcitrans, given the abundance of substrates such as straw and sugarcane bagasse, filter cake and vinasse, which, together, favor the occurrence of fly population outbreaks. Barros et al. (2010) reported an outbreak of stable flies in the state of Mato Grosso do Sul, where the authors associated the large numbers of flies with vinasse used in the irrigation of sugarcane plants.
A class of agents with potential for use in biological control are entomopathogenic nematodes (EPNs), which are obligate parasites of soil-dwelling insects (Kaya & Gaugler, 1993). The importance of EPN as biological control agents has been revealed in the results of several studies published in recent decades, which describe their pathogenic effect on various types of pests. However, in Brazil, the use of EPN has been studied mainly in agriculture, and the number of researches evaluating its action on pests of veterinary importance is still scanty. In response to the heightened resistance of insect pests to chemical pesticides (Barros et al., 2019), mainly due to the excessive use, research has focused on different microorganisms over the years, seeking to identify their pathogenic potential on different parasites of economic importance, and their feasibility for use in biological control or application in integrated control (chemical and biological).
The purpose of this study was to determine the effectiveness of the EPNs Heterorhabditis bacteriophora HP88 and H. baujardi LPP7 against S. calcitrans larvae after being exposed to byproducts of the sugar and alcohol industry, and the response of EPNs when exposed to vinasse at different temperatures (investigate whether high vinasse temperatures can cause any risk to EPNs) and their action on S. calcitrans larvae of different ages.

Material and Methods
The S. calcitrans colony used in this study was raised on a laboratory benchtop (27±1° C and 70-80% relative humidity -RH) using the methods described by Mello (1989) and Moraes (2007). The EPNs colonies followed the method described by Lindegren et al. (1993), and it was maintained and multiplied via in vivo multiplication in Galleria mellonella (Lepidoptera: Pyralidae). Infective juveniles (IJs) were stored in a Biochemical Oxygen Demand (BOD) temperature-controlled incubator (Eletrolab®, model EL 202/4) at 16 ± 1° C and 70-80% RH in a 40 mL cell culture flask for less than 7 days. To calculate the levels used in this study, IJs were counted in twelve 10 μL aliquots taken from an aqueous EPNs suspension. After counting the IJs in the 12 aliquots, the highest and lowest number of EPNs/aliquot were discarded and the average number of IJs in the remaining 10 aliquots was calculated. Based on this calculation, the concentration of the suspensions was adjusted to IJs/mL (Taylor et al., 1998).

Efficacy of EPNs against S. calcitrans larvae in vinasse
EPNs were quantified and adjusted to a concentration of 2000 IJs/ml, and aliquots were extracted and placed in containers with the following concentrations of vinasse: 0% (control group, distilled water only), 50% and 100%, kept in BOD incubators (Eletrolab®, model EL 212) at temperatures of 16 °C (control), 25 °C, and 35 °C. The containers were taken out of the incubators after 72h, and aliquots were analyzed under an optical microscope to determine the effects caused by temperature and vinasse on EPNs viability.
Seventy-two hours after the beginning of the viability bioassay, surviving nematodes from each concentration/ temperature were used in a subsequent bioassay to evaluate their efficacy to stable fly larvae in Petri dishes.
Aliquots were extracted from each group and quantified at 400 viable IJs/larva of S. calcitrans. Six-day-old S. calcitrans larvae were subjected to mortality tests involving five larvae per Petri dish (nine centimeters in diameter). Petri dishes were kept on shelves in the laboratory at 27±1 °C and 70±10% RH. The experiment was conducted in an entirely randomized design with six repetitions. The bioassay was monitored daily for seven days. S. calcitrans larvae mortality data were subjected to the Shapiro-Wilk normality test. After detecting normality of the distribution, an analysis of variance (three-way ANOVA) was applied, and if the groups showed a significant effect, the means were compared using Tukey's test. In all analyses, a global significance level of 5% was adopted. All the analyses were performed using Prisma GraphPad version 9.1.2 software.

Bioassay II
Efficacy of EPNs on S. calcitrans larvae of different ages in filter cake Groups of five 4, 6 and 8-day-old larvae were placed on 9-cm diameter Petri dishes (lined with filter paper) containing 2.5 grams of filter cake, and exposed to a concentration of 400 IJs/larva of S. calcitrans (diluted in 4 mL of distilled water). The volume of water added to the control groups was the same as the experimental groups, but without EPNs. The dishes were kept on shelves in the laboratory at 27±1 °C and 70±10% RH. The experiment was performed in an entirely randomized design with six repetitions. The treatment groups resulted from the 2x3 factorial design of two EPNs species (H. bacteriophora HP88 and H. baujardi LPP7) and three ages of larvae (four, six and eight days) developing in filter cake. The bioassay was monitored daily for seven days. S. calcitrans larvae mortality data were subjected to the Shapiro-Wilk normality test. After detecting normality of the distribution, an analysis of variance (two-way ANOVA) was applied, and if the groups showed a significant effect, the means were compared using Tukey's test. In all analyses, a global significance level of 5% was adopted. All the analyses were performed using Prisma GraphPad version 9.1.2 software.

Bioassay III
Efficacy of different concentrations of EPNs on S. calcitrans larvae in sugarcane bagasse Groups of five 6-day-old larvae were placed on Petri dishes (9 cm diameter, lined with filter paper) containing three grams of chopped sugarcane bagasse, to which were added 100, 300 and 500 IJs/larva of S. calcitrans diluted in 4 ml of distilled water. The volume of water added to the control groups was the same as the experimental groups, but without EPNs. The plates were kept on shelves in the laboratory at 27±1 °C and 70±10% RH. The experiment was carried out in an entirely randomized design, with seven treatment groups subjected to six repetitions. Efficacy of H. bacteriophora HP88 and H. baujardi LPP7 on stable fly larvae was evaluated in sugarcane bagasse. The bioassay was monitored daily for seven days. S. calcitrans larvae mortality data were subjected to the Shapiro-Wilk normality test. After detecting normality of the distribution, an analysis of variance (one-way ANOVA) was applied, and if the groups showed a significant effect, the means were compared using Tukey's test. In all analyses, a global significance level of 5% was adopted. All the analyses were performed using Prisma GraphPad version 9.1.2 software.

Results and Discussion
Efficacy of EPNs against S. calcitrans larvae A significant effect was observed in the in the EPNs vs. vinasse and EPNs vs. temperature interactions, indicating that the lethality of the nematodes evaluated varied as a function of vinasse concentrations and storage temperature. There was no significant effect of the triple interaction of the tested factors. The EPN H. bacteriophora HP88 showed higher efficacy than H. baujardi LPP7 at all temperatures, especially at 50% and 100% vinasse, with more pronounced mortality at 35°C at 100% vinasse concentration (Table 1). When the efficacy of EPNs was evaluated as a function of increasing temperature, the virulence of H. bacteriophora HP88 did not decrease at all vinasse concentrations. However, the virulence of H. baujardi LPP7 decreased as the temperature increased, especially at concentrations of 50% and 100% vinasse ( Table 1).
Vinasse had no negative effect on the virulence of H. bacteriophora HP88, regardless of the temperature applied, since the mortality rate of fly larvae was always greater than 86% in all concentrations of vinasse subjected to all temperatures. For H. baujardi LPP7, there was no statistical difference in larval mortality when this EPN was applied at 50% vinasse compared to without vinasse. However, when subjected to 100% vinasse, at all tested temperatures, this EPN decreased its virulence, since larval mortality was lower, especially at 25° and 35 °C ( Table 1).
Overall, H. bacteriophora HP88 showed higher mortality rates than H. baujardi LPP7 against S. calcitrans larvae, and its virulence was not affected by increasing of temperature and vinasse concentration. Conversely, the virulence of H. baujardi LPP7 declined with increasing of temperature and vinasse concentrations, indicating that this EPN is more sensitive to these conditions than H. bacteriophora HP88.
The conditions of this bioassay provided some of the most important information of the data set obtained from the present study, because, considering the possibility of creating an integrated stable fly control system using EPNs, the temperature that these organisms can withstand is a crucial factor for the success of such an application. These data indicate that IJs of H. baujardi LPP7 are highly vulnerable to both pure vinasse and temperatures close to the limit for stable fly development (Florez-Cuadros et al., 2019), while H. bacteriophora HP88 proved to be little affected. Studies dealing with the heat resistance of the genus Heterorhabditis indicate that, in general, the optimal temperature for the development, infection, and reproduction of these EPNs is around 25 °C (Shaurub et al., 2015). However, several publications reported the adaptation of EPNs species to temperatures around 35 °C (Kusakabe et al., 2019). Similarly, the considerable efficacy of EPNs for pest control has been confirmed even when exposed to high temperatures, and on a wide variety of hosts (Mendonça et al., 2019). In the specific case of H. baujardi LPP7, an optimal temperature of 28 °C for replication and infection has been reported, with emphasis on the origin of this strain, namely, the Amazon rainforest; hence, it is probably already adapted to tropical environments (Dolinski et al., 2007). González (2006) reported the viability of H. baujardi LPP7 at temperatures of 35 °C for two hours, with no loss in efficacy against G. mellonella larvae. In the present study, H. baujardi LPP7 lost efficacy against S. calcitrans larvae after 72 h (in a different substrate) at 35°C. Furthermore, it should be noted that this EPN has been evaluated on different hosts and that G. mellonella showed considerably greater sensitivity to this EPN than the stable fly.
The resistance of these EPNs to solutions containing 50% vinasse, and the short time required to reach high mortality peaks at this concentration, suggest that EPNs could be used in combination with the fertigation of sugarcane fields with vinasse, since this is the vinasse concentration usually applied (Rossetto, 1987). Corrêa et al. (2013) described the storage of vinasse in pre-fertigation tanks, which may also be an approach for the application of EPNs, or even the addition of EPNs in hydrants near sprinklers, where the temperature should be even lower, and occasionally in places previously identified with high numbers of S. calcitrans larvae. It should be noted that despite the possible high productivity (flies/area) in filter cake, the size of areas under fertigation is a more significant factor (Corrêa et al., 2013). Therefore, vinasse plus mulch is the most important substrate associated with the occurrence of S. calcitrans outbreaks (Corrêa et al., 2013), since it attracts stable flies and hastens the process of sugarcane straw decomposition (Souza et al., 2021). and 'y' comparing each EPN within the vinasse concentrations (x > y).

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Efficacy of nematodes on stable fly larvae Efficacy of EPNs on S. calcitrans larvae of different ages in filter cake The age of stable fly larvae did not affect their mortality rates due to EPNs. Both H. bacteriophora HP88 and H. baujardi LPP7 showed higher larvae mortality than the control group ( Table 2). Not many experiments are reported in the literature regarding the interaction between filter cake and insect pests that develop in this substrate. Monteiro et al. (2016), using a similar methodology with lower concentrations of H. bacteriophora HP88,i.e,25,50,100,150 and 200 IJs/larva of S. calcitrans at eight days of age. The larvae mortality rate reported by the above-mentioned authors was 76.7% with 150 IJs/larva and 83.3% when applying 200 IJs/larva. The high mortality of stable fly larvae of different ages by H. bacteriophora HP88 in the present study agrees with the previous findings of Monteiro et al. (2016).
The larvae mortality rate achieved in the present study with H. bacteriophora HP88 was lower (83.33%) than the 96.7% mortality reported by Leal et al. (2017) using 200 IJs/larva. However, in the bioassay developed by the cited authors, EPNs were not exposed to substrates that could influence their effectiveness. Hence, it is reasonable to infer that filter cake had a limited impact on the action of H. bacteriophora HP88 in the present study. This byproduct of the sugar and ethanol industry may interfere more significantly in the virulence of H. baujardi LPP7, since Leal et al. (2017) reported that the proportion of 200 IJs/larva of S. calcitrans succeeded in causing a 93.3% mortality rate.
An important factor for the occurrence of population outbreaks of this fly in Brazil is the huge volume of byproducts generated by the sugarcane industry and the large quantities left behind on the field after harvesting. Therefore, it is worth considering that the area needed for the application of EPNs in filter cake under natural conditions would be drastically smaller if compared to the area of straw that remains on the ground after the sugarcane harvest, since the filter cake is stored in windrows (a more limited area). Furthermore, the heat produced by filter cake fermentation reaches 35.5 ± 5.8°C (Corrêa et al., 2013) limiting distribution of stable fly larvae to windrow edges. These areas are less hot and more humid, also facilitating the movement of EPNs in their search for stable fly larvae.

Efficacy of different concentrations of EPNs on S. calcitrans larvae in sugarcane bagasse
The EPN H. bacteriophora HP88 caused higher mortality rates than the control group at all the tested concentrations. The concentration that caused the highest mean mortality rate was 500 IJs/larva, which was significantly higher than the concentrations of 100 and 300 IJs/larva; the latter showed no statistically significant difference (Figure 1). It was noted that the more than 60% mortality rate of larvae treated with 500 IJs of H. baujardi LPP7, which is higher than that obtained in other biological pest control studies (Moraes et al., 2008;Alves et al., 2012). Of particular importance for control systems using fungi is that certain antifungal and antibacterial substances are released by the larval microbiota of S. calcitrans (Dillon & Dillon, 2004;Moraes et al., 2014). Given that the pathogenicity of EPNs is caused by septicemia after invasion and secretion of bacteria directly inside the host, it is possible that these nematode species are not directly affected by the larval microbiota of the dipteran, which explains the difference commonly reported in the mortality rates of immature stages of S. calcitrans caused by these two control methods.
A comparison of the results of this bioassay with the findings reported by Leal et al. (2017) using the same concentration of 100 IJs/larva revealed a loss of efficacy of H. bacteriophora HP88 in the order of 30%. In the case of H. baujardi LPP7, this comparison reveals a lower mortality rate of 53.4%, with the caveat that the method employed by Leal et al. (2017) did not use materials such as sugarcane on the infection dishes, indicating that the decrease in treatment efficacy (100 IJs/larva) is likely associated with the presence of sugarcane bagasse.

Conclusions
The results found in the present study suggest that EPNs can be a potential component in integrated strategies of stable fly control and outbreak prevention in areas of sugar and alcohol production.